Automated Staining and Decolorization of Biological Material

ABSTRACT

An improved method and apparatus for staining samples of biological material for accurate analysis of the sample. Biological material is applied to a substrate, such as a microscope slide. The biological specimen is then stained with a selected staining composition, which may be gentian violet for a Gram&#39;s Stain analysis. The stained biological material is at least partially decolorized and the level of decolorization is analyzed optically. If necessary, the decolorizing step and the optical analysis steps are repeated until a selected level of decolorization is obtained.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application which claims priority from U.S.patent application Ser. No. 10/274,613, filed Oct. 21, 2002, entitledAUTOMATED STAINING AND DECOLORIZATION OF BIOLOGICAL MATERIAL, which is adivisional of U.S. Pat. No. 6,468,764, issued Oct. 22, 2002, whichclaimed priority to U.S. Provisional Application No. 60/116,485, filedJan. 20, 1999, all of the same title and inventors.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods and apparatus for thediagnostic staining of biological material. More particularly, thepresent invention relates to methods and apparatus for stainingbiological material on microscope slides in an automated fashion.

2. Background and Summary of Prior Art

Biological material, whether viruses, bacteria, or various smears orsamples of blood, mucus, and the like, have been analyzed for centuries,since Van Leeuvenhoek invented the microscope. Typically the sample ofmaterial is applied to a microscope slide, stained or otherwise renderedinto an analyzable state, and analyzed by a human technician orscientist.

As laboratory services for hospitals, physician's offices,veterinarians, and other life-science-based enterprises becomeincreasingly “outsourced,” the laboratory's ability to analyze slides ofbiological material rapidly and accurately becomes increasinglyimportant. Several steps have been made toward automating theseprocesses, but it seems unlikely that the skilled human technician willbe almost entirely removed from the process in the foreseeable future.

A fairly typical and important laboratory staining technique is known asGram's Stain, which was devised by H. C. J. Gram. The Gram's Stain is a“gateway” test that indicates to the technician the presence (orabsence) of certain bacteria in a sample of biological material andgives the technician or scientist information necessary or helpful tomake further analysis. For instance, Gram's Stain can be used todetermine which pathogens are suspected and lead to an antibioticprescription until further identification can be conducted.

Like several staining or analysis methods, Gram's Stain involvestreating the biological material, usually applied to a conventionalmicroscope slide, with a number of reagents or stains. The reagents orstains emphasize or highlight the presence (or absence) of certain typesor features of bacteria or other biological material that is helpful tothe technician. In the Gram's Stain, the bacteria are treated first withgentian violet, and then with a formulation of iodine conventionallyknown as Gram's iodine. This stains almost all of the bacteria a deepblue or violet. “Gram positive” bacteria absorb the gentian violet andGram's iodine into their cellular structure, while “Gram negative”bacteria are stained only superficially. The sample is then washed withacid alcohol, which “decolorizes” or washes the color from Gram negativebacteria. Thus, when adequate decolorization has occurred, the blue orviolet Gram positive bacteria can be distinguished from the colorless(or less deeply blue or violet) Gram negative bacteria. A“counter-stain,” of fuchsine for example, may be applied to turn theblue or violet bacteria to a reddish shade to improve their visibility.Decolorization is critical to the Gram's Stain because too littledecolorization can yield false Gram positives and too muchdecolorization can yield false Gram negatives.

Several past attempts at providing automating the slide preparation andstaining process have met with limited success. For example, U.S. Pat.No. 4,029,470, Jun. 14, 1977 to Wilkins et al. provides an apparatus forautomatically staining a single microscope slide without a labtechnician touching the slide. This patent addresses the decolorizationissue by timing the application of decolorizing agent in selectedvolume. The time/volume control of decolorization is insufficient toaccurately decolorize a Gram Stain. The decolorization process is simplytoo dependent upon observation and manual work to be so easilycontrolled.

GG&B Company of Wichita Falls, Texas, sells a more sophisticated slidestainer under the registered trademark Quick Slide ° . This device fullyautomates the preparation of slides for analysis and is a useful tool inthe modern medical laboratory. Nevertheless, the Quick Slide® device isnot capable of accurately decolorizing slides for a staining processsuch as the Gram's Stain.

U.S. Pat. Nos. 5,545,535; 4,665, 024; and 4,639,421 all discloseflourescent gram stains and methods of analyzing bacteria stained withthe flourescent dyes in which a spectral analysis of the fluorescence ofthe stained bacteria is used to analyze the Gram positive and negativebacteria in the sample. These inventions do not address thedecolorization issue because it seems that decolorization is lessimportant (or unimportant) where flourescent dyes or stains are used inlieu of the conventional Gram's Stain of gentian violet.

A need exists, therefore, for an automated method and apparatus forstaining biological material and accurately decolorizing the stainedsample prior to analysis.

SUMMARY OF THE INVENTION

It is a general object of the present invention to provide an improvedmethod and apparatus for staining samples of biological material foraccurate analysis of the sample. These and other objects of theinvention are achieved by applying the biological material to asubstrate, preferably a microscope slide. The biological material isthen stained with a selected staining composition, which may be gentianviolet for a Gram's Stain analysis. The stained biological material isat least partially decolorized and the level of decolorization isanalyzed optically. If necessary, the decolorizing step and the opticalanalysis steps are repeated until a selected level of decolorization isobtained.

Other objects, features, and advantages of the present invention willbecome apparent with reference to the drawings and description, whichfollow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of the apparatus according to thepresent invention.

FIG. 2 is a high-level flow chart of the method according to the presentinvention.

FIG. 3 is a schematic depiction of the decolorization analysis module ofthe apparatus of FIG. 1.

FIG. 4 is a more detailed schematic description of an apparatus forstaining biological materials according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the figures, and particularly to FIG. 1, a high-levelflowchart depicts the steps of the method or process according to thepresent invention. At block 11, a sample of biological material isapplied to the substrate, typically a conventional microscope slide, tocreate a specimen. The biological specimen can be affixed or applied ina number of conventional ways, which depend largely on preference andsubsequent processing. The biological specimen may include blood, mucus,tissue samples, or the like, and is obtained in conventional ways.

At step 13, the biological specimen is stained or dyed according to theanalytical process to be employed. For the Gram Stain, the dye or stainis crystal or gentian violet in combination with Gram's iodine. Thestain or dye stains the components of the biological specimen, in somecases only superficially; in others, the dye is absorbed within the cellstructure.

At step 15, the biological specimen is decolorized, that is, the dye orstain that is not absorbed within the cell structure, but onlysuperficially, is washed away. For the Gram Stain, the decolorization isaccomplished by washing the specimen with acid alcohol (alcohol treatedto be slightly acidic). The washing may be controlled by volume of fluidapplied and time of application to yield a very rough measure of thelevel of decolorization achieved in this step.

At step 17, the level of decolorization is measured or analyzed. Thespecific method of analysis employed is discussed in greater detail withreference to FIG. 3. Generally, the method applies radiation (visiblespectrum, infrared, ultraviolet) to the “run-off” fluid or materialresulting from application of acid alcohol to the specimen. Theradiation passes through the acid alcohol, which itself is stained withgentian violet, Gram's iodine, and a safrin counter-stain. Because therun-off fluid is stained, it absorbs certain wavelengths or frequenciesof the radiation and transmits others. The radiation transmitted throughthe run-off fluid is measured and compared to a threshold value. Thethreshold value is empirically determined based upon the characteristicsof the staining and decolorization process used (e.g. dye or staincharacteristics and composition and decolorizing agent characteristicsand composition).

At step 19, the comparison is analyzed to determine if the desired orselected level of decolorization has occurred. If so, the process ends,at step 21, and the slide is ready for further processing or analysis.If the level of decolorization is inadequate, more washing withdecolorant occurs, as well as the analysis of step 17, until the sampleis adequately decolorized.

FIG. 2 is a schematic depiction of the apparatus employing the processof FIG. 1. The apparatus comprises a slide carrier or mount 31 on whichthe microscope slide or other specimen substrate is placed or carried. Acollector 33 is provided to collect or catch process fluids as they runoff the slide. The apparatus is also provided with five reservoirs, 35,37, 39, 41, 43 for the process fluids, which, for the Gram Stainprocess, include crystal or gentian violet, Gram's iodine, safrin(counter-stain), acid alcohol, and water. These reservoirs are connectedto a fluid handling and metering system 45 that washes the biologicalspecimen with the appropriate fluid in the appropriate amount. Time- andvolume-controlled application of the staining fluids (violet, iodine,and safrin) is adequate for those fluids to perform their function. Aspreviously discussed, time and volume control of the decolorizationprocess, alone, is not adequate.

FIG. 3 is a schematic representation of the “decolorization module” ofthe apparatus of FIG. 2. The decolorization module can be manufacturedas part of the slide carrier or mount (31 in FIG. 2), or can be aphysically separate portion of the apparatus. The run-off fluid from thedecolorization process, which includes acid alcohol, either diluted withwater or not, is collected by collector 33. The run-off from thedecolorization step is collected in a chamber 51, which is preferablyoptically transparent. In addition to chamber 51, the decolorizationmodule includes a source of radiation 53 arranged and located toirradiate the fluid contents of chamber 51 with radiation of a selectedwavelength or combination of wavelengths.

The run-off fluid, being stained, will transmit certain wavelengths ofradiation and absorb others. The radiation transmitted through chamber51 and run-off fluid impinges on a photo detector 55. Photo detector 55and associated electrical circuitry, including filters and the like,generates an electrical signal whose strength is proportional to theintensity and wavelength of the radiation impinging on detector 55. Thissignal is input to a comparator or similar device capable of comparingthe signal to a pre-determined threshold value representative ofadequate decolorization. If the signal compares favorably with thethreshold value, the user is alerted and the stained slide is indicatedto be ready for visual analysis. If the measured level of decolorizationindicates that decolorization is inadequate, the specimen can be washedagain and the run-off fluid analyzed as set forth above.

The transmissibility of radiation through the run-off fluid is afunction of the level of decolorization of the specimen on the slide.Thus, the level of decolorization can be empirically determined tocorrespond to a threshold value of transmitted radiation detected byphoto detector 55. Thus, the proper level of decolorization is opticallydetected without human interference or involvement, which permitsaccurate, automated slide staining to a degree not previously available.

FIG. 4 is a more detailed schematic of the apparatus according to thepresent invention. Central to the apparatus is the control systemresiding on a personal computer (PC) 111, which may be a PC dedicatedfor use with the invention, or a conventional PC with control softwareresident on the hard drive and RAM, for use with the invention asneeded. The other components, which may be external to the PC, arehoused within an enclosure (not shown) that is coupled to the PC.

The enclosure includes reservoirs (not shown) for the process fluids,which include, for Gram staining, gentian violet, Gram's iodine,distilled water, safrin, and acid alcohol. Other fluids may beappropriate for other staining processes. An EEPROM 113, programmedappropriately, is coupled to the computer through an IEEE 488 interface,and also to a series of stepper motors 115. Together, EEPROM 113 andstepper motors 115 control the flow of process fluids from thereservoirs, through fluid passages, to the microscope slide. The run-offof decolorization fluid (acid alcohol), is captured temporarily in aglass cuvette 121. EEPROM 113 is an appropriately programmedBell-Milgray 93LC46/P. Stepper motors 115 and their associatedcontrollers are Oriental Motors Model PK264-01A, and Newark NDN2878U,respectively.

Adjacent the microscope slide, a regulated power supply powers a lightsource 123, which is a Gilway 5-Volt, 775-Ampere technical lamp. This isthe radiation source and is arranged to direct visible-spectrumradiation through the cuvette 121 and its contents. A Digi-KeyOPT202P-ND photodiode 125 with an on-chip amplifier is arranged on theside of cuvette 121 opposite the lamp. A wavelength-selective filter127, Omega Optical 200 BP25, is interposed between the run-off fluid inthe cuvette 121 and photodiode 125, and absorbs or filters radiationfrom the lamp 123 outside the spectral range of about 200 to 340nanometers, which permits passage of the violet portion of the visiblespectrum. The violet spectral components passed by filter 127 areabsorbed (or not) by the run-off or decolorizing fluid in the cuvette121 and the total passed radiation impinges on the photodiode 125. Theuse of filter 127 reduces the number of wavelengths of radiationimpinging on photodetector 125 and reduces the likelihood of error inthe detection of the amount of radiation passing through the run-offfluid, which is indicative of the level of decolorization.

The electrical signal output of photodiode 125, which is proportional tothe level of decolorization of the slide, is routed through an AnalogDevices AD976AAN analog-to-digital converter 131. The digital output ofconverter then is input to the computer for analysis by a comparisonroutine. The comparison routine may compare the output of the photodiodeto a discrete, empirically determined threshold, or may use a variety ofconventional, but more sophisticated, analysis techniques to determinewhether the stained sample is appropriately decolorized or whether moredecolorization (acid alcohol rinse) is warranted. The PC can displayvarious information about the staining process, including process fluidlevels, number of slides processed, decolorization level, calibrationdata, and the like. The above-described embodiment is by way of exampleof a staining apparatus according to the present invention for Gramstaining, it is not intended to be limiting of the invention.

The present invention provides a number of advantages. Primarily, itpermits automation of the preparation of stained slides when thestaining process involves decolorization, as do many slide stainingprocedures, including the Gram Stain. The method and apparatus accordingto the present invention are sufficiently simple to be reliable andrelatively inexpensive to produce.

The invention has been described with reference to a preferredembodiment thereof. It is thus not limited, but is susceptible tovariation and modification without departing from the scope of theclaims.

1. (canceled)
 2. An apparatus for detecting the level of staining in amicroscope slide, the apparatus comprising: a plurality of fluidreservoirs, each containing a fluid, the fluids including at least astain fluid and a decolorizer fluid, a slide handling mechanismconfigured to position and support a microscope slide with a biologicalspecimen thereon for application of fluids from the fluid reservoir forstaining of the biological specimen; a controller coupled to the fluidreservoirs to control flow of the fluids onto the biological specimen onthe microscope slide; a collector configured to at least temporarilycollect the decolorizer fluid that runs off the biological specimen; anelectro-optical detector coupled to the controller and arranged todetect a level of decolorization in the decolorizer fluid collected inthe collector by comparing a characteristic of the collected decolorizerfluid to a selected threshold that indicates an adequate level ofstaining in the biological specimen, the detector including: a source ofradiation arranged to irradiate the collector and the collecteddecolorizer fluid with radiation of at least one selected wavelengththat is absorbed by the stain fluid contained in the collecteddecolorizer fluid; a radiation detector configured to detect a degree ofabsorption of the radiation of selected wavelength by the collecteddecolorizer fluid, the detector coupled to the controller for comparisonof the degree of absorption with the selected threshold; and awavelength-selective filter between the source of radiation and theradiation detector.
 3. The apparatus according to claim 2, wherein thestain fluid comprises Gentian violet and iodine.
 4. The apparatusaccording to claim 2, wherein the decolorizer fluid is acid alcohol. 5.The apparatus according to claim 2, wherein the selected wavelength ofthe radiation is that corresponding to a violet portion of the visiblespectrum.
 6. The apparatus according to claim 2, wherein the radiationdetector is a photodiode.
 7. A decolorization module for use in anapparatus for automatically staining a biological specimen on amicroscope slide, the apparatus including a plurality of fluidreservoirs, including at least a reservoir of stain fluid and areservoir of decolorizer, the decolorization module comprising: a slidehandling mechanism including a microscope slide carrier configured tosupport the microscope slide with the biological specimen duringapplication of fluid from the fluid reservoirs; a controller coupled tothe fluid reservoirs to control dispensation of at least the stain fluidand the decolorizer onto the biological specimen on the microscope slideon the slide carrier; a collector configured to at least temporarilycollect the decolorizer that runs off the biological specimen; anelectro-optical detector coupled to the controller and arranged todetect a level of decolorization in the decolorizer collected in thecollector by comparing a characteristic of the collected decolorizer toa selected threshold that indicates an adequate level of decolorizationin the biological specimen, the detector including: a source ofradiation arranged to irradiate the collected decolorizer with radiationof at least one selected wavelength that is absorbed by the stain fluidcontained in the collected decolorizer; a radiation detector configuredto detect a degree of absorption of the radiation of selected wavelengthby the collected decolorizer, the detector coupled to the controller forcomparison of the degree of absorption with the selected threshold; anda wavelength-selective filter between the source of radiation and thedetector.
 8. The apparatus according to claim 7, wherein the stain fluidcomprises Gentian violet and iodine, and each is contained in a separatefluid reservoir.
 9. The apparatus according to claim 7, wherein thedecolorizer is acid alcohol.
 10. The apparatus according to claim 7,wherein the selected wavelength of the radiation is that correspondingto a violet portion of the visible spectrum.
 11. The apparatus accordingto claim 8, wherein the radiation detector is a photodiode.